PP2A Type Phosphatases in Sea Urchin Eggs

Abstract

Four peaks of the p-nitrophenyl phosphate (pNPP) splitting activity were obtained by QAEToyopearl chromatography in the extract of sea urchin eggs from homogenate in 0.2M KCl solution containing 0.1% Triton X100 and so on. In 2 among these 4 peaks, pNPP splitting reactions were strongly inhibited by 10nM okadaic acid (OA) and calyculin A (CLA), potent and specific inhibitors of protein phosphatase 2A (PP2A). High sensitivities of pNPP splitting reaction to OA and CLA in these 2 peaks suggest that pNPP splitting results from the reaction catalyzed by PP2A, which reaction is expected to play roles in gene expression, signal transduction and cell movement such as cell division. OA sensitive pNPP splitting activities in these 2 peaks were eluted by FPLC gel permeation chromatography (Superdex 200HR) with approximate molecular mass of 160 kDa, corresponding to that of PP2A trimeric holoenzyme in mammalian cells. By immunoblot analyses with anti-human PP2A catalytic subunit antibody, an immunoreactive 36 kDa protein was found by SDS-PAGE in a peak of OA-sensitive pNPP splitting activity, obtained by QAE-Toyopearl chromatography. Sea urchin eggs have at least 2 PP2A like enzymes with similar molecular masses to that of mammalian PP2A and one of them contains human type catalytic subunit.